We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding (G) proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA and downregulates protein synthesis in virally infected cells), and PERK (activated under conditions of ER stress). In order to subvert the anti-viral defense mediated by PKR viruses produce inhibitors of the kinase. Several members of the poxvirus family express two different types of PKR inhibitors: a pseudosubstrate inhibitor (such as the vaccinia virus K3L protein that resembles the N-terminal third of eIF2alpha) and a double-stranded RNA binding protein called E3L. High-level expression of human PKR inhibited the growth of yeast, and co-expression of the vaccinia virus K3L protein or the related variola (smallpox) virus C3L protein restored yeast cell growth. We identified 12 PKR mutations that confer resistance to K3L inhibition both in yeast and in vitro. In vitro studies revealed that WT PKR and the PKR mutants phosphorylated eIF2alpha with the same kinetics;however, the mutant kinase was less sensitive to inhibition by K3L. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity. Our results support the identification of the eIF2alpha binding site on an extensive face of the C-terminal lobe of the kinase domain and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We proposed that these paradoxical effects on pseudosubstrate versus substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser51 phosphorylation site. Phylogenetic analyses of the eIF2alpha kinases plus four unrelated protein kinases revealed fast evolution of the PKR kinase domain in vertebrates. These evolutionary studies also revealed evidence of positive diversifying selection at specific sites in the PKR kinase domain. Substitution of positively selected residues in human PKR with residues found in other species altered the sensitivity to PKR inhibitors from different poxviruses. Comparing the sensitivity of human and mouse PKR to poxviral pseudosubstrate inhibitors revealed differences that were traced to positively selected residues near the eIF2alpha-binding site. Interestingly, 10 of the 12 mutations identified in the genetic screen for PKR mutations conferring resistance to K3L inhibition occurred at sites that were under positive selection during evolution. Taken together, our results indicate how an antiviral protein (PKR) evolved to evade viral inhibition while maintaining its primary function (phosphorylation of eIF2alpha). Moreover, our identification of species-specific differences in PKR susceptibility to viral inhibitors has important implications for studying human infections in nonhuman model systems. The translation initiation factor eIF2 binds the initiator Met-tRNA to the small ribosomal subunit. To gain further insights into the role of GTP binding and hydrolysis by eIF2, we mutated a conserved residue in the eIF2gamma GTP-binding domain Switch I element. The mutation impaired Met-tRNA binding and enhanced initiation from a non-canonical UUG codon. Second site suppressors of the Switch I element mutation restored Met-tRNA binding but had differing impacts on UUG initiation. This uncoupling of start codon selection and Met-tRNA binding affinity to eIF2 indicates a more direct role for eIF2 in start site recognition than previously appreciated. Interestingly, overexpression of eIF1, which is thought to monitor codon-anticodon interaction during translation initiation, suppressed initiation at UUG codons in the eIF2gamma mutants. We propose that structural alterations in eIF2gamma subtly alter the conformation of Met-tRNA on the 40S subunit and thereby affect the fidelity of start codon recognition independent of Met-tRNA binding affinity. The GTP-binding protein eIF5B catalyzes ribosomal subunit joining in the final step of translation initiation. Our previous studies revealed that GTP hydrolysis by eIF5B activates a regulatory switch required for eIF5B release from the ribosome following subunit joining. Mutations in eIF5B that impair GTP hydrolysis impair yeast cell growth due to failure to dissociate the factor from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA within the body of the small ribosomal subunit as well as mutations in domain II of eIF5B suppressed the toxic effects of the GTPase-deficient mutants of eIF5B. Interestingly, hydroxyl radical mapping experiments revealed that domain II of eIF5B docks on the ribosome in the vicinity of helix h5. Additional studies showed that helix h5 is also important for the function of the translation elongation factor GTP-binding protein eEF2. Thus, these studies provide in vivo evidence supporting a functionally important docking of domain II of translational GTPases on the body of the small ribosomal subunit. The translation factor eIF5A, the sole protein containing the unusual amino acid hypusine N&#949;-(4-amino-2-hydroxybutyl)lysine, was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation. Hypusine is required for eIF5A to associate with ribosomes, and to stimulate methionyl-puromycin synthesis. As eIF5A did not stimulate earlier steps of translation initiation, and depletion of eIF5A in yeast only modestly impaired protein synthesis, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular mRNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay and to nucleocytoplasmic transport. Using molecular genetic and biochemical studies, we recently showed that eIF5A promotes translation elongation. Depletion of eIF5A or shifting a temperature-sensitive (ts-) eIF5A mutant to the non-permissive temperature resulted in the accumulation of polysomes, mimicking the affect of the translation elongation inhibitor cycloheximide. Moreover, inactivation of eIF5A increased the ribosomal transit time, the amount of time required for a ribosome following initiation to synthesize and release a completed protein. The translation elongation defect in extracts from the eIF5A ts- mutant strain was suppressed by addition of recombinant eIF5A from yeast, but not by a derivative lacking hypusine. Moreover, eIF5A enhanced the rate of tripeptide synthesis in reconstituted translation elongation assays. Finally, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin and impaired programmed ribosomal frameshifting. These results indicate that eIF5A might function together with eEF2 to promote ribosomal translocation. As eIF5A is a structural homolog of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor.